Composition containing mixed extract of mulberry and poria cocos bark for preventing, improving or treating neurodegenerative disorders

ABSTRACT

The present invention relates to a composition comprising the mixed extract of mulberry and Poria cocos peel for preventing, improving or treating neurodegenerative disorders. The mixed extract of mulberry and Poria cocos peel which is the active ingredients contained in the composition of the present invention, has a memory improving activity through inhibiting of acetylcholine esterase and a neuroprotective effects and neuron protection by inhibiting the formation of beta amyloid and tau phosphorylation and promoting NGF production. Thus, the present invention may be useful as a pharmaceutical composition for preventing or treating degenerative neurological diseases, or as a health food for the above purpose.

TECHNICAL FIELD

The present invention relates to a composition comprising the mixedextract of mulberry and Poria cocos peel for preventing, improving ortreating neurodegenerative disorders.

RELATED ART

Degenerative neurological disease is a disease in which mental functionis deteriorated due to gradual structural and functional loss ofneurons. Degenerative neurologic disease may be accompanied by symptomssuch as dementia, extrapyramidal abnormality, cerebellar abnormality,sensory disturbance, and movement disorder due to nerve celldegeneration in specific areas of the nervous system. In addition,complex symptoms may occur due to multiple abnormalities at the sametime. The disease is diagnosed according to the clinical manifestationof the patient. However, the symptoms vary widely, and differentdiseases often have common clinical symptoms, making diagnosis bedifficult (Soc. Sci. Med. Vol. 40. No. 6, pp. 847-858, 1995).

Degenerative neurological disease is a symptom of the disease slowlydevelops, and is often caused by aging. Once the disease has developed,the disease progresses continuously for several years or decades untildeath, and the fundamental treatment is difficult, so the social burdenis great. It is known that genetic influences such as family history arevery likely to be the cause of the disease, but acquired factors alsoplay an important role. Degenerative neurological disorders can beclassified according to their clinical symptoms, such as progressivedementia (Alzheimer's disease, etc.), neurological disorders (such aspick disease), posture and movement disorders (such as Parkinson'sdisease), progressive ataxia, muscular atrophy and weakness, sensory andmotor disorders and so on (International Journal of Engineering andTechnology, Vol. 2, No. 4, August 2010 Classification ofNeurodegenerative Disorders Based on Major Risk Factors EmployingMachine Learning Techniques).

Cytotoxicity of Aβ plaque and tangles of hyper-phosphorylated tau isattracting attention as a direct cause of Alzheimer's dementia, atypical degenerative brain disease.

Aβ is produced from the precursor APP and is produced by the action ofenzymes such as β-secretase and γ-secretase, and is distributed outsidethe nerve cells. When the Aβ concentration is low, it is in a dissolvedstate, but when it is above a certain level, the Aβ proteins bind toeach other to form an insoluble senile plaque. This substance candestroy peripheral nerve cells by causing inflammation andneurotoxicity. For example, neuronal death and microgranulosis observedin patients with Alzheimer's disease is thought to be associated withthe senile plaque. In vitro test results showed that Aβ could induceactivation of microglial cells (brain macrophages). This supports thehypothesis that microgranulosis and brain inflammation found in thebrains of patients with Alzheimer's disease is a major cause ofAβ-induced Alzheimer's disease. To date, there have been no widelyaccepted therapies or therapies that are expected to remarkably dissolveAβ once the senile plaque is formed, or to prevent the formation ofdeposits.

The tau consists of four parts: the N-terminal protruding part, theproline aggregation domain, the micro-organelle binding domain and theC-terminal (Mandelkow et al., Acta. Neuropathol., 103, 26-35, 1996). Itplays a role in connecting the microtubules that form the physicalstructure of nerve cells. It is known that degenerative brain diseasessuch as tauopathy are caused by abnormally hyperphosphorylated ortransformed tau in the nervous cells of the central nervous systemAlzheimer's disease, Picks disease, Frontotemporal dementia andparkinsonism linked to chromosome 17(FTDP-17) and so on, are typicaltauopathies (Lee et al., Annu. Rev. Neurosci., 24, 1121-1159, 2001;Bergeron et al., J. Neuropathol. Exp. Neurol., 56, 726-734, 1997;Bugiani et al., J. Neuropathol. Exp. Neurol., 58, 667-677, 1999;Delacourte et al., Ann. Neurol., 43, 193-204, 1998; Ittner and Gotz,Nat. Rev. Neurosci., 12, 65-72, 2011).

In the 1980s Alzheimer's study, neurotrophic factors were suggested tohave therapeutic potential for degenerative neurological disorders(Nature. 1987 Sep. 3-9; 329(6134):65-8. Amelioration of cholinergicneuron atrophy and spatial memory impairment in aged rats by nervegrowth factor). Studies have shown that cholinergic neurons in the basalforebrain, lost due to aging, which is known to be one of the causes ofAlzheimer's disease, are restored by administering nerve growth factor(NGF) to the lateral ventricle, resulting in an improvement in memorycapacity of experimental animals. So, studies have been continuing totreat degenerative neurological diseases using neurotrophic factors. Ina subsequent study, a study was carried out to restore motor nervefunction by injecting Brain-derived neurotrophic factor (BDNF),Neurotrophin-3(NT-3), Neurotrophin-4(NT-4), and ciliary neurotrophicfactor (CNTF), which are neurotrophic factor family into experimentalanimals that impaired motor nerve function by nodulating the facialnerve and sciatic nerve, and positive results were obtained (Nature.1992 Dec. 24-31; 360(6406):757-9. Brain-derived neurotrophic factorprevents the death of motoneurons in newborn rats after nerve section.).Furthermore, in the experiment using a recombinant mouse (wobbler) witha disease in which the number and function of motor neurons weregradually lost as the aging progressed, the number of motor neurons wasincreased and the function was improved by administering BDNF and CNTFto the mouse (Science. 1994 Aug. 19; 265(5175):1107-10. Arrest of motorneuron disease in wobbler mice cotreated with CNTF and BDNF). Inaddition, to the above-mentioned studies, it has been reported thatneurotrophic factors improve memory, cognition, and behavioral disordersin experimental animals by increasing the number and function of neuronsin various sensory and motor neuropathic models.

The scientific name of the mulberry is Morus alba (

), and when the fruit of the deciduous mulberry tree is magenta, it ispicked, dried and used as a medicinal herb. In oriental medicine, it isknown to be used for treating dizziness, tinnitus, thirsty and diabetesand so on. In Japan, it has the effect of treating Yanghyulguopoong andWind-Heat, and is known to be applied to treatments such as tonic, painmedication, insomnia, tinnitus, dizziness, back pain, and constipationand so on (Namba, T., The Encyclopedia of Wakan-Yaku with Color PicturesVol. 1 Hoikusha, Osaka, Japan, 1993). In Donguibogam, mulberry treatsthirst and benefits five organs, and mulberry has mulberry tree's vitalforces(

) (Donguibogam korean translation committee, Donguibogam, Namsandang,Seoul, Korea, 2000). In Chinese pharmacopoeia (CP), it is alsoprescribed medicines for the treatment of dizziness, tinnitus, insomniaand thirst etc.

Mulberry has anthocyanin, phenolic acid and flavonoid and so on, as maincomponents and it has been reported that the isolated single componentand its extract have blood glucose lowering, MAO (monoamine oxidase)inhibitory activity, antioxidative effect and neuronal cell protectioneffect.

The scientific name of Bokryeong (

) is Poria cocos. Poria cocos is a plant of the class familyPolyporaceae, in the order Aphyllophorales, in the classEubasidiomycetes, Hymenomycetidae. Poria cocos is a kind of fungusbelonging to bacillus, which is a brown rot fungus, which is a kind ofsaprophyte organism in the pine tree, but it is also parasitic to theroot of living pine tree. Mycelium grows while branching to white, andhyphae start to grow together. When the proper environmental conditionssuch as temperature and humidity are continued, hard lumps of sclerotiaare formed. This is called bokryeong. According to the color of theinside, white is Poria cocos wolf, rose pink is Jeokbokryeong. The outershell of Poria cocos is called Poria cocos peel.

The main components of the Poria cocos are Parchymic acid (C₃₃H₅₂O₅),Pinicolic acid (C₃₀H₁₈O₃), 3β-Hyderoxy-lanosta-7.9(11), 24-Trien-21-oicacid, Tumulosic acid (C₃₁H₅₀O), Ebricoic acid (C₃₁H₅₀O₃) and the like.It also contains purgiminic acid, ebricoic acid, Polypotenic acid A, Cand triterpenoids etc. And it also contains ergosterol, lecithin,adenine, choline, glucose, fructose and protein, and a large amount ofinorganic substances.

Poria cocos is sweet and the nature of Poria cocos is warm, and itstaste and properties are all light, so it is said to be a medicinalsubstance with cool properties. In the past literature, it is describedthat the Poria cocos can relieve the thirst, discharge the urinesmoothly, remove the moisture, moderate the body condition, and enhanceenergy by harmonizing the function of digestive system, by smoothing thewaist, the circulation of abdominal blood eases well. It was mainly usedfor prescription related to memory. In recent articles, anti-diabeticand anticancer effects of Poria cocos have been reported (Ling Hui,Evaluation of the chemotherapeutic and chemopreventive potential oftriterpenoids from Poria cocos, Ling Hui, doctoral thesis of departmentof pharmacy, National University of Singapore, 2010).

Poria cocos peel is the outer shell of Poria cocos sclerotium belongingto the polyporaceae. It is known as a medicinal substance that has theeffect to moderate edema by releasing water. Especially, it containsabundantly lorostane-based triterpene poricoic acid, and Donguibogamstates that the Poria cocos peel is used as a component ofOpisan(tangerine peel, arrecae pericarpium, ginger peel, morus albapeel) prescribed for edema. In recent studies, it has been reported thatPoria cocos peel have therapeutic effects such as diuretic, urinarystimulation, and edema reduction etc. (Triterpenes from the surfacelayer of Poria cocos, Takaaki tai, Phytochem, vol. 39, no. 5, 1995;Urinary metabonomic study of the surface layer of Poria cocos as aneffective treatment for chronic renal injury in rats, ying yong zhao etal, Journal of Ethnopharmacology, vol. 148, no. 2, s2013).

With this background, the inventors have conducted studies to developmaterials for pharmaceutical composition and food composition for theeffective prevention, improvement and treatment of degenerativeneurological diseases. As a result, the inventors of the presentinvention confirmed that these herbal extracts exhibit significantmemory recovery activity in a brain neuropathy model induced by variousbrain injury or memory-inhibiting drugs during a neuropsychologicalactivity study of the mulberry and Poria cocos peel. In addition, theseherbal extracts have been shown to inhibit the production of substancesthat cause neuronal cell death in the brain and to protect the nervecells by promoting the expression of proteins promoting neural cellregeneration and differentiation. The present inventors have completedthe present invention by confirming that when the mulberry and Poriacocos peel herbal extracts are mixed at a certain ratio, they shows amaximum effect, compared to the herbal extract as a single component.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

It is an object of the present invention to provide a pharmaceuticalcomposition for preventing and treating of degenerative neurologicaldiseases and a health functional food for preventing and ameliorating ofdegenerative neurological diseases, which contains a mixed extract ofmulberry and Poria cocos peel as an active ingredient.

Technical Solution

In order to achieve the above object, the present invention provides apharmaceutical composition for preventing and treating of degenerativeneurological diseases, and a health functional food for preventing andameliorating of degenerative neurological diseases, which are containinga mixed extract of mulberry and Poria cocos peel.

Advantageous Effect

According of the present invention, the mixed extract of mulberry andPoria cocos peel inhibits the production of amyloid-β (Abeta) andphosphorylation of tau which may cause degenerative brain diseases byinducing neuronal death, and exhibit an effect of protecting nerve cellsby promoting the NGF production, which is a factor promoting a neuralcell survival and differentiation. In addition, it exhibits an effect ofinhibiting acetylcholine esterase (AChE) to increase nerve conductionand to mediate memory enhancing action. Therefore, the mixed extract ofmulberry and Poria cocos peel can be effectively used for thedevelopment of pharmaceutical composition for preventing and treating ofdegenerative neurological diseases including dementia or for thedevelopment of health food for preventing and ameliorating ofdegenerative neurological diseases.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results obtained by injecting a herbal mixed extract, asingle extract, and a control drug in a model inducing neurotoxicitywith beta amyloid, and staining with NeuN, a neural cell marker.

FIG. 2 shows the results obtained a mixed extract, a single extract, anda control drug to a model induced by neurotoxicity with beta amyloid,and staining with FJB, a cell death marker.

MODE OF THE INVENTION

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition forpreventing or treating of degenerative neurological diseases, comprisinga mixed extract of mulberry and Poria cocos peel as an activeingredient.

The mixed extract of the present invention can be provided according toa conventional method for preparing an herbal extract, but specificallymeans a crude extract obtained by using water, alcohol, or a mixture ofwater and alcohol. The alcohol which is one of the extraction solventsmay be, but is not limited to, a lower alcohol having 1 to 4 carbonatoms, preferably methanol, ethanol, butanol or alcohol spirit, morepreferably ethanol. Here, the alcohol spirit means ethanol produced byfermenting starch raw material or saccharide raw material and distillingit. Further, the mixed solvent of water and alcohol is not particularlylimited, and may be mixed at any desired ratio.

In one embodiment of the present invention, the mixed extract of themulberry and Poria cocos peel may be extracted with 60 to 80% (v/v)ethanol, 65 to 75 (v/v) % ethanol or 70 (v/v) %, but is not limitedthereto.

In another embodiment of the present invention, the weight ratio of themulberry and Poria cocos peel may be 4 to 7:1, 4 to 6:1 or 5:1, but isnot limited thereto.

The mixed extract of the present invention includes all the forms of anextract obtained by extracting a mixture of mulberry and Poria cocos andan extract obtained by separately extracting mulberry and Poria cocosseparately and then mixing each extract. Specifically, mulberry andPoria cocos peel are mixed and extracted at the weight ratio, ormulberry and Poria cocos peel are respectively extracted by appropriatemethods, and then each extract is mixed at the weight ratio.

More specifically, the mixed extract of the present invention can beobtained as follows. First, the mulberry and Poria cocos peel are washedand dried, respectively, and then the dried herbs are cut to obtain thecut herbs. The dried cut of mulberry and Poria cocos peel are mixed at aweight ratio of 0.1 to 10 times as the weight of the Poria cocos peel,and preferably, the mixture is prepared by mixing at a weight ratio ofmulberry:Poria cocos peel=4 to 7:1. Water, lower alcohol having 1 to 4carbon atoms or a mixture of water and the lower alcohol, preferably70(v/v) % ethanol, is added to the mixture of mulberry and Poria cocospeel in an amount of 1 to 20(v/w) times, or preferably 5 to 10(v/w)times as the weight of the mixture. Then, the mixture is subjected toextraction at a temperature of 10° C. to 100° C., preferably at roomtemperature for 1 hour to 72 hours, preferably 48 hours according tocold water extraction, hot water extraction, ultrasonic extraction,reflux cooling extraction, heat extraction, or supercritical extraction.Preferably, the extraction can be performed by cold water extractiononce, and concentration under reduced pressure to produce a mixedextract of mulberry and Poria cocos peel.

The composition containing the mixed extract of mulberry and Poria cocospeel according to the present invention as an active ingredient can beused for the treatment of degenerative neurological diseases.Accordingly, the present invention provides a pharmaceutical compositionfor preventing and treating degenerative neurological diseases, whichcomprises a mixed extract of mulberry and Poria cocos peel as an activeingredient and an use of a mixed extract of mulberry and Poria cocospeel for the preparation of a therapeutic agent for degenerativeneurological diseases, and a method of treating a degenerativeneurological disease comprising administering to a subject atherapeutically effective amount of the mixed extract of a mulberry andPoria cocos peel.

As described above, the degenerative neurological disease according tothe present invention means a degenerative disease of mental functioncaused by gradual structural and functional loss of nerve cells(neurons). Specifically, it includes diseases selected from the groupconsisting of Alzheimer's disease, Creutzfeldt-Jakob disease,Huntington's disease, multiple sclerosis, Guilin-Barre syndrome,Parkinson's disease, Lou Gehrig's disease, progressive dementia causedby gradual neuronal death and progressive ataxia and so on.

In one embodiment of the present invention, the pharmaceuticalcomposition for preventing and treating of degenerative neurologicaldiseases containing the mixed extract of mulberry and Poria cocos peelas an active ingredient at an amount of 0.01 to 90 parts by weight, 0.1to 90 parts by weight, 1 to 90 parts by weight, or 10 to 90 parts byweight, based on 100 parts by weight of the total pharmaceuticalcomposition, but is not limited thereto, and may vary depending on thecondition and the type and progress of disease.

In another embodiment of the present invention, the pharmaceuticalcomposition for preventing and treating degenerative neurologicaldiseases containing the mixed extract of mulberry and Poria cocos peelas an active ingredient may be formulated into pharmaceuticalpreparations for the prevention and treatment of degenerativeneurological disorders, including pharmaceutically acceptable carriers,diluents or excipients.

Examples of the carrier, excipient and diluent include lactose,dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol,starch, acacia rubber, alginate, gelatin, calcium phosphate, calciumsilicate, cellulose, methylcellulose, microcrystalline cellulose,polyvinylpyrrolidone, water, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition of the present invention may be formulated in the formof oral preparations such as powders, granules, tablets, capsules,suspensions, emulsions, syrups, aerosols and a form of externalpreparation, suppository or sterilized injection solution in accordancewith a conventional method in order to prepare them in the form ofmedicines. In general, in the case of formulation, it can be prepared byusing a diluent such as filler, an extender, a binder, a wetting agent,a disintegrant, a surfactant, or an excipient which is usually used.Solid form preparations for oral administration include tablets, pills,powders, granules, capsules and the like, which may contain at least oneexcipient such as starch, calcium carbonate, sucrose, lactose, gelatinand the like. In addition to simple excipients, lubricants such asmagnesium stearate and talc may also be used. Liquid preparations fororal use include suspensions, solutions, emulsions, and syrups. Inaddition to commonly used simple diluents such as water and liquidparaffin, various excipients such as humectants, sweeteners, fragrances,preservatives and the like may be included. Formulations for parenteraladministration include sterilized aqueous solutions, non-aqueoussolutions, suspensions, emulsions, freeze-dried preparations andsuppositories. Examples of the suspending agent include propyleneglycol, polyethylene glycol, vegetable oil such as olive oil, injectableester such as ethyl oleate, and the like. Examples of the suppositorybase include witepsol, macrogol, tween 61, cacao butter, laurinum, andglycerogelatin and so on.

The present invention provides a method for treating a neurodegenerativedisease, which comprises administering a pharmaceutical compositioncontaining the mixed extract of the mulberry and Poria cocos peel as anactive ingredient to a mammal, including a human, in a therapeuticallyeffective amount.

The dosage of the pharmaceutical composition for the prevention andtreatment of degenerative neurological diseases containing the mixedextract of mulberry and Poria cocos peel as an active ingredient of thepresent invention may vary depending on the age, sex, and body weight ofthe patient, Generally, an amount of 0.1 to 100 mg/kg, preferably 1 to30 mg/kg, may be administered once to several times per day. The dosagemay also be increased or decreased depending on the route ofadministration, degree of disease, sex, body weight, age, healthcondition, diet, administration time, administration method, excretionrate and the like. Accordingly, the dose is not intended to limit thescope of the invention in any way.

The pharmaceutical composition containing the mixed extract of mulberryand Poria cocos peel of the present invention can be administered invarious routes to mammals such as rats, mice, livestock, and humans. Allmodes of administration may be expected, for example, by oral, rectal orintravenous, intramuscular, subcutaneous, intra-uterine dural orintracerebral injection.

The mixed extract of mulberry and Poria cocos peel in accordance withthe present invention has little toxicity and side effects, andtherefore can be safely used for long-term use for preventive purposes.

The present invention also provides a food composition comprising themixed extract of mulberry and Poria cocos peel. The mixed extract ofmulberry and Poria cocos peel inhibits the production of substancescausing neuronal cell death in the brain and promote the expression ofproteins promoting neural cell regeneration and differentiation toprotect nerve cells and can be effectively used for the production ofhealth functional food and general food which can exhibit the effect ofpreventing and improving neurodegenerative diseases in particular.

Specifically, the health functional food defined in the presentinvention is defined by the “Act on Health Functional Foods 2002” newlydefined as “the functional and safety of the human body has beensufficiently established and the Food and Drug Administration Notice2004-12 Of the Regulation on the Recognition of Ingredients of HealthFunctional Foods or Ingredients prescribed in the Act”.

The food composition containing the mixed extract of mulberry and Poriacocos peel according to the present invention can be used variously forfood for symptom relief of degenerative neurological diseases. As a foodto which the herbal extract of the present invention can be added,various foods such as beverages, gums, tea, vitamin complex, healthfunctional food, and health functional beverage and they can be used inthe form of pills, powders, granules, infusions, tablets, capsules orbeverages.

The amount of the mixed extract of mulberry and Poria cocos peel in thefood of the present invention is generally 0.1 to 15% by weight,preferably 0.2 to 10 wt %, and in the case of the health functionalbeverage composition, 0.1 to 30 g, preferably 0.2 to 5 g, based on 100ml, may be included.

When the food composition according to the present invention is preparedin a beverage form, there are no particular restrictions on the liquidingredients other than those containing the herbal extracts as essentialingredients in the proportions indicated. In addition, various flavoringagents or natural carbohydrates may be added as an additionalingredient, such as ordinary beverages.

Examples of the above-mentioned natural carbohydrates includemonosaccharides such as glucose and fructose; Disaccharides such asmaltose, sucrose; Polysaccharides such as Dextrin, cyclodextrin and thelike, or sugar alcohols such as xylitol, sorbitol and erythritol and soon. As natural flavors other than those mentioned above, naturalflavoring agents (thaumatin, stevia extract (e.g., rebaudioside A,glycyrrhizin etc.)) and synthetic flavors (saccharin, aspartame, etc.)can be advantageously used. The ratio of the natural carbohydrate isgenerally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of thetotal composition of the present invention.

In addition to the above, the food composition of the present inventionmay further contain various additives such as various nutrients,vitamins, minerals (electrolytes), flavorings such as synthetic flavorsand natural flavors, colorants and thickeners, pectic acid and itssalts, alginic acid and its salts, organic acids, protective colloidthickeners, pH adjusting agents, stabilizers, preservatives, glycerin,alcohols, carbonating agents used in carbonated drinks, and the like. Inaddition, the compositions of the present invention may contain naturalfruit juice and pulp for the production of fruit juice drinks andvegetable drinks. These components can be used independently or incombination. The ratio of such additives is not particularly limited,but is generally selected in the range of 0 to about 20 parts by weightper 100 parts by weight of the total food composition of the presentinvention. In another embodiment, the present invention is a method forpreventing, ameliorating or treating a degenerative neurologicaldisease, comprising administering a composition containing an activeingredient of a combination of the mixed extract of mulberry and Poriacocos peel. Alternatively, the present invention provides a use of themixed extract of mulberry and Poria cocos peel for preventing,ameliorating or treating a degenerative neurological disease. The dose,dosage form, administration method, etc. of the composition containingmulberry and Poria cocos peel mixed extract for such method or use areas mentioned above.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be explained in detail with reference to thefollowing examples and experiments. However, the following examples andexperiments are only to illustrate the present invention, and the scopeof the present invention is not limited thereto.

<Example 1> Preparation of Poria cocos Peel Extract According to thePresent Invention

Dried mulberry and Poria cocos peel were purchased on herbal medicalstore in Kyungdong market and contaminants were removed and well-driedherbal was used for the experiment. Mulberry and Poria cocos peel cutwith a cutting machine were mixed at a weight ratio of 5:1 in an amountof 300 g. And 3 L of 70 (v/v) % aqueous ethanol solution was added tothe herbal mixture and the mixture was cooled at room temperature for 48hours. After filtration, the mixture was concentrated under reducedpressure, and then lyophilized to obtain a crude herbal extract (crudeextract) to give Example 1 (See Table 1).

TABLE 1 Preparation of Poria cocos peel extract according to the presentinvention Preparation of poria cocos peel extract Raw material accordingamount (g) to the Poria Amount present cocos Solvent of ExtractionExtraction Product Yield invention Mulberry peel selection solventtemperature time (g) (%) Example 1 250 50 70% EtOH 3 L Room 2 days 19.756.58 temperature

<Comparative Example 1> Preparation of Mulberry Extract

200 g of the same botanical raw material mulberry as Example 1 wasprepared according to the same method of Example 1 to obtain a crudeextract of Mulberry, which was used as Comparative Example 1 (See Table2).

<Comparative Example 2> Preparation of Poria cocos Peel Extract

200 g of the same botanical raw material of Poria cocos peel as Example1 was prepared according to the same method of Example 1 to obtain acrude extract of Poria cocos peel as Comparative Example 2 (see Table2).

TABLE 2 Yield of single extracts of mulberry and Poria cocos peel Rawmaterial amount (g) Poria cocos Solvent Solvent Extraction ExtractionProduct Yield Mulberry peel type amount temperature time (g) (%)Comparative 200 — 70% EtOH 2 L Room 2 days 8.4 4.2 Example 1 temperatureComparative — 200 70% EtOH 2 L Room 2 days 6.8 3.4 Example 2 temperature

<Comparative Examples 3 to 8> Preparation of Mixed Crude Extracts ofMulberry and Poria cocos Peel

The same botanical raw material as the mulberry and Poria cocos peelused in Example 1 was used in the experiment. Mulberry and Poria cocospeel cut with a cutting machine, Mulberry and Poria cocos peel weremixed as shown in Table 3, and then 70% ethanol aqueous solution havinga volume of 10 times of the herbal mixture volume was added to theherbal mixture, and the mixture was cold-extracted at room temperaturefor 48 hours, After concentration under reduced pressure, the mixturewas lyophilized to obtain a crude herbal extract (crude extract), whichwas used as Comparative Examples 3 to 8.

TABLE 3 Yield of mixed herbal extracts of mulberry and Poria cocos peelRaw material amount (g) Poria Amount cocos Solvent of ExtractionExtraction Product Yield Mulberry peel selection solvent temperaturetime (g) (%) Comparative 100 100 70%   2 L Room 2 days 16.96 8.48Example 3 EtOH temperature Comparative 150 50 70%   2 L Room 2 days15.78 7.89 Example 4 EtOH temperature Comparative 200 20 70% 2.2 L Room2 days 14.39 6.54 Example 5 EtOH temperature Comparative 50 150 70%   2L Room 2 days 14.20 7.10 Example 6 EtOH temperature Comparative 40 20070% 2.4 L Room 2 days 18.94 7.89 Example 7 EtOH temperature Comparative20 200 70% 2.2 L Room 2 days 19.76 8.98 Example 8 EtOH temperature

<Experimental Example 1> Test of Memory Improvement and Nerve CellProtection Effect of the Mixed Herb Extracts

<1-1> Preparation of Experimental Animals

Scopolamine Induced Memory Impairment Model

Six-week-old ICR mice were used for the experiment and each experimentalgroup consisted of 8 animals.

After 30 minutes of the Conducting drug (Example), Comparative drug(comparative example) and Donepezil (Control drug) oral administration,1 mg/kg scopolamine was intraperitoneally administered. Normal animalswere injected with the same amount of physiological saline. Behavioralstudies were performed after 30 minutes of scopolamine administration.

Aβ Infusion Model

Six-week-old ICR mice were used in the experiment. The animals wereanesthetized, fixed in a stereotaxic apparatus (Stoelting) and injectedwith 3 μl of vehicle (artificial CSF) or Aβ₄₂ for 6 minutes using aHamilton micro syringe (fitted with a 26-gauge needle).

Animals treated with Aβ₄₂ were randomly divided into experimentalgroups. From 2 days after administration of Aβ₄₂, the experimental drugand the positive control drug were administered once a day for 11 days.

The lyophilized drug, comparative drug and control drug were suspendedin 3% HPMC aqueous solution, and all the drugs were prepared on the dayof the experiment.

Measurement of Protein in Brain

To examine the efficacy of mixed herbal extracts on the production ofnerve cell death inducer or substances involved in cell protection, Sixweeks old ICR mouse were administered the experimental drug, thecomparative drug and the control drug for 5 days. On the 5th day 4 hoursafter the administration, Experimental animals were sacrificed and thebrain was separated. Parts except cerebellum and Medulla oblongata wereused in the experiment.

<1-2> Passive Avoidance Task

Passive avoidance experiments were performed in two independentlyseparated bright and dark square boxes. Bright areas (20×20×20 cm) wereilluminated with 50 W incandescent lamps. Light and dark areas (20×20×20cm) were spaced 1 cm apart and 2 mm stainless steel rods were installed.

In the habituation phase, the two sections were separated by aguillotine door (5×5 cm), and the guillotine door was opened after 30seconds after placing the experimental animals in the light section. Theanimals were allowed to freely navigate. When they reached the darkarea, the guillotine door was closed and the animals were taken outafter 3 seconds.

Acquisition trials were conducted 24 hours later. After 30 minutes ofdrug administration, 1 mg/kg of scopolamine was intraperitoneallyadministered and 30 minutes later, the behavioral experiment wasperformed. The experimental mice were initially placed in the brightzone and the door between the zones was opened after 30 seconds. Whenthe experimental mice entered the dark area, the guillotine door wasclosed, and an electrical foot shock was immediately applied through astainless steel rod for 2 seconds at 0.5 mA intensity, and the animalswere taken out after 10 seconds. The time that the animals entered thedark zone was measured.

After performing the acquisition trials and giving a 24-hour restperiod, the experimental animals were placed in a bright area formeasuring the retention trial and the time until entering the dark areawas measured. Both the acquisition trial and the retention trial wereperformed to determine the time taken for the mouse to enter all fourfeet in a dark room from a bright room.

As a result, as shown in Tables 4 and 5 below, the memory improvementeffect was confirmed in each of the memory loss model by scopolamine andthe Aβ₄₂-induced Alzheimer's dementia induction model. Compared withComparative Examples 1 and 2, which are sole extracts of mulberry andPoria cocos peel, and Comparative Examples 3 to 8, which are mixedextracts of mulberry and Poria cocos peel, mulberry, Poria cocos peel5:1 mixture showed significant spatial memory enhancement effect. Themixed extract of Example 1 showed an efficacy equal to or higher than 1mg/kg of donepezil (DPZ), which is known as an acetylcholinesteraseinhibitor.

TABLE 4 Scopolamine model - memory enhancing efficacy in passiveavoidance experiment Total weight Raw material comparison amount (g)Mulberry: Poria Poria cocos Acqui- Reten- Mul- cocos peel Volume sitiontion berry peel weight ratio (mg/kg) time time Normal 62.8 270.3 Vehicle72.3 92.3 example 1 250 50 5:1 100 59.4 255.9 Comparative 200 — Mulberryalone 100 88.8 110.4 Example 1 Comparative — 200 Poria cocos 100 77.5187.2 Example 2 peel alone Comparative 100 100 1:1 100 69.3 163.2Example 3 Comparative 150 50 3:1 100 64.2 189.9 Example 4 Comparative200 20 10:1  100 58.7 172.8 Example 5 Comparative 50 150 1:3 100 66.1151.8 Example 6 Comparative 40 200 1:5 100 83.4 157.3 Example 7Comparative 20 200  1:10 100 59.2 168.6 Example 8 Donepezil 1 77.4 243.5

TABLE 5 Aβ₄₂ administration model - memory enhancing efficacy in passiveavoidance experiment Total weight Raw material comparison amount (kg)Mulberry: Poria Poria cocos Acqui- Reten- Mul- cocos peel Volume sitiontion berry peel weight ratio (mg/kg) time time Normal 52.4 285.3 Vehicle66.9 99.9 example 1 250 50 5:1 100 71.3 269.7 Comparative 200 — Mulberry100 49.8 152.4 Example 1 alone Comparative — 200 Poria cocos 100 41.2138.9 Example 2 peel alone Comparative 100 100 1:1 100 39.8 163.2Example 3 Comparative 150 50 3:1 100 57.4 188.9 Example 4 Comparative200 20 10:1  100 62.5 162.2 Example 5 Comparative 50 150 1:3 100 51.3143.1 Example 6 Comparative 40 200 1:5 100 46.9 127.6 Example 7Comparative 20 200  1:10 100 38.6 142.9 Example 8 Donepezil 1 78.4 244.2

<1-3> Y-Maze Test

The Y-maze experiment was conducted to investigate the effect ofshort-term memory. The animals were placed on one arm of the Y-maze andallowed to move freely for 8 minutes. Spontaneous alternation and totalnumber of entries were measured. When the animals were consecutivelyentered into three arms, they were defined as alternation entry, and theratio was calculated by the following equation 1.

Spontaneous alternation ratio (%)=[(number of changes)/(total number ofarm entry−2)]×100  [Equation 1]

As a result, as shown in Tables 6 and 7, we confirmed the memoryimprovement effect in the memory decline model by scopolamine andAβ₄₂-induced Alzheimer's dementia induction model. Particularly, incomparison with Comparative Examples 1 and 2, which are the soleextracts of mulberry and Poria cocos peel, and Comparative Examples 3 to8, which are mixed extracts of mulberry and Poria cocos peel, 5:1mixture of mulberry and Poria cocos peel according to Example 1 showedsignificant cognitive and memory enhancing effect. In particular, themixed extract of Example 1 showed better efficacy than Donepezil (DPZ) 1mg/kg, which is known as the acetyl cholinesterase inhibitor.

TABLE 6 Scopolamine model - memory-enhancing efficacy in Y-mazeexperiment Total weight Raw material comparison amount (kg) Mulberry:Poria Poria cocos Total spontaneous cocos peel Volume number Truealternation Mulberry peel weight ratio (mg/kg) of entries enter (%)Normal 42 32 76.2 Vehicle 51 27 52.9 example 1 250 50 5:1 100 50 36 72.0Comparative 200 — Mulberry 100 40 23 57.5 Example 1 alone Comparative —200 Poria cocos 100 41 26 63.4 Example 2 peel alone Comparative 100 1001:1 100 48 29 60.4 Example 3 Comparative 150 50 3:1 100 35 23 65.7Example 4 Comparative 200 20 10:1  100 40 25 62.5 Example 5 Comparative50 150 1:3 100 33 19 57.6 Example 6 Comparative 40 200 1:5 100 59 3355.9 Example 7 Comparative 20 200  1:10 100 42 27 64.3 Example 8Donepezil 1 64 43 67.2

TABLE 7 Aβ₄₂ administration model - memory enhancing efficacy in Y-mazeexperiment Total weight Raw material comparison amount (kg) Mulberry:Poria Poria cocos Total spontaneous cocas peel Volume number Truealternation Mulberry peel weight ratio (mg/kg) of entries enter (%)Normal — — — — 37 29 78.4 Vehicle — — — — 38 19 50.0 example 1 250 505:1 100 59 45 76.3 Comparative 200 — Mulberry alone 100 45 28 62.2Example 1 Comparative — 200 Poria cocos 100 42 28 66.7 Example 2 peelalone Comparative 100 100 1:1 100 36 22 61.1 Example 3 Comparative 15050 3:1 100 51 35 68.6 Example 4 Comparative 200 20 10:1  100 42 25 59.5Example 5 Comparative 50 150 1:3 100 33 20 60.6 Example 6 Comparative 40200 1:5 100 58 35 60.3 Example 7 Comparative 20 200  1:10 100 44 24 54.5Example 8 Donepezil — — — 1 61 43 70.5

<1-4> Novel Object Recognition (NOR) Task

Inside a black box of 45 cm width, 45 cm length, 50 cm height, twoplastic objects of the same size but different shapes were placed. Theexperimental animals were allowed to move freely for 8 minutes and thetime spent on each object was measured (training session) 24 hourslater, the experimental animals were placed in a box with one familiarobject and one new object in the training session and the time spent ineach object was measured (test session). The recorded values werecalculated using the following equation (2), which is expressed as arecognition result of a new object.

Recognition rate of new objects (%)=Time spent on new objects/(Timespent on new objects+Time spent on experienced objects)×100  [Equation2]

As a result, as shown in Tables 8 and 9, we confirmed the memoryimprovement effect in the each of the scopolamine-induced memory declinemodels and the Aβ₄₂-induced Alzheimer's models. Compared withComparative Examples 1 and 2, which are sole extracts of mulberry andPoria cocos peel, and Comparative Examples 3 to 8, which are mixedextracts of mulberry and Poria cocos peel, mulberry, Poria cocos peel5:1 mixture showed significant cognitive and memory enhancement.Compared with Donepezil (DPZ) 1 mg/kg, which is known as the acetylcholinesterase inhibitor, the mixed extract of Example 1 showed theequivalent cognitive and memory improving effect in the scopolamineinduced memory formation inhibition model, and exhibited a cognitive andmemory improvement effect equal to or higher than that of the Aβ₄₂administration model.

TABLE 8 Scopolamine Model - Memory Enhancement Effect in NOR ExperimentTotal weight Raw material comparison amount (kg) Mulberry: Familiar Newobject Poria Poria cocos New object object Total object recognitioncocas peel Volume recognition recognition recognition time Mulberry peelweight ratio (mg/kg) time time time ratio (%) Normal — — — — 13.7 5.319.0 72.1 Vehicle — — — — 11.6 13.9 25.5 45.5 example 1 250 50 5:1 1007.4 3.9 11.3 70.2 Comparative 200 — Mulberry 100 3.8 5.6 9.4 50.4Example 1 Comparative — 200 Poria cocos 100 11.2 8.3 19.5 64.2 Example 2peel Comparative 100 100 1:1 100 16.6 12.7 29.3 56.7 Example 3Comparative 150 50 3:1 100 12.3 11.6 23.9 61.5 Example 4 Comparative 20020 10:1  100 11.3 12.3 23.6 57.9 Example 5 Comparative 50 150 1:3 10013.0 11.5 24.5 53.1 Example 6 Comparative 40 200 1:5 100 11.1 9.8 20.957.1 Example 7 Comparative 20 200  1:10 100 15.3 13.9 29.2 61.4 Example8 Donepezil — — — 1 12.9 7.9 20.8 68.0

TABLE 9 Aβ₄₂ administration model - memory enhancing efficacy in NORexperiment Total weight Raw material comparison amount (kg) Mulberry:New object Poria Poria cocos New object New object Total objectrecognition cocas peel Volume recognition recognition recognition ontime Mulberry peel weight ratio (mg/kg) on time on time on time ratio(%) Normal — — — — 19.7 5.3 25.0 78.8 Vehicle — — — — 12.6 13.9 26.547.5 example 1 250 50 5:1 100 13.4 3.9 17.3 77.5 Comparative 200 —Mulberry 100 9.8 5.6 15.4 63.6 Example 1 Comparative — 200 Poria cocos100 17.2 8.3 25.5 67.5 Example 2 peel Comparative 100 100 1:1 100 22.612.7 35.3 64.0 Example 3 Comparative 150 50 3:1 100 18.3 11.6 29.9 61.2Example 4 Comparative 200 20 10:1  100 14.3 12.3 26.6 53.8 Example 5Comparative 50 150 1:3 100 16.0 11.5 27.5 58.2 Example 6 Comparative 40200 1:5 100 17.1 9.8 26.9 63.6 Example 7 Comparative 20 200  1:10 10021.3 13.9 35.2 60.5 Example 8 Donepezil — — — 1 18.9 7.9 26.8 70.5

<1-5> Immunohistochemical Staining

For immunohistochemical staining, the Aβ infusion animal described inExperimental Example 1-1 was perfused with 1×PBS (phosphate bufferedsaline), fixed with 4% paraformaldehyde, and brain was extracted. Thiswas fixed in the same solution for one day and stored in a 30% sucrosesolution and the solution was changed every two days until frozen at 4°C. After that, brain tissue was frozen sufficiently at −20° C. bydropping OCT (optimal cutting temperature) compound in a cryostat, andthen made into a 30 μm-thick section, and stored at 4° C. in apreservative solution. Immunohistochemical staining was performed withthe hippocampus portion. The tissues washed with PBS were treated with1% H₂O₂ for 15 minutes, After that the tissues were treated with 0.05 MPBS, 1.5% normal goat serum, 0.5 mg/ml bovine serum albumin, 0.3% tritonX-100 and goat NeuN primary antibody (1:500) and were reacted at 4° C.for 24 hours to prevent nonspecific reactions. After the primaryantibody was removed, the tissue was reacted with aperoxidase-conjugated secondary antibody (1:200) for 90 minutes, and theABC was diluted in the buffer and allowed to react at room temperaturefor about 1 hour. After washing three times with PBS, the tissue wasdeveloped with 0.02% DAB and 0.01% H₂O₂ and subjected to ethanol andxylene dehydration to prepare slide samples.

For FJB (Fluoro-Jade-B) staining, tissue sections were fixed with PBScontaining 4% paraformaldehyde for 5 minutes and stored at −70° C. Thenext day, the slides dried for 3 hours were immersed in a 0.06%permanganate potassium nitrate solution for 10 minutes. After rinsingwith water, the slides were soaked in 0.1% acetic acid and 0.0004% FJBsolution (Calbiochem, San Diego, Calif., USA) for 20 minutes. The slideswere washed three times with distilled water and dried at 55° C. for 10minutes. A multifocal microscope (Olympus, Japan) was used for imagingand the photographs were taken with a soft imaging system video camera.

As a result, as shown in FIG. 1, NeuN expression as a marker of neuroncells was higher than that of the control group by administration ofmixed extract of botanical raw materials. This means that the reductionof neuronal cells was suppressed in the herbal mixed extractadministration group. The extracts of mulberry and Poria cocos peelalone had a protective effect on neurons but were weaker in efficacythan herbal mixed extracts. Donepezil, the control drug, failed to showneuroprotective effects.

As shown in FIG. 2, the expression of FJB, a marker of apoptosis, wasdecreased by the herbal mixed extract as compared with the control. Thisresult implies that the increase in the number of nerve cells identifiedin FIG. 1 is mediated through inhibition of neuronal cell death inducedby aggregation of beta amyloids.

<1-6> Measurement of Beta Amyloid Production in Brain

ELISA experiments were conducted to investigate the inhibitory effect ofherbal mixed extracts on beta amyloid production, which is known to be amajor cause of Alzheimer's disease.

The brain of normal animals in Experimental Example <1-1> was extracted,and then the hippocampal region was separated, and RIPA buffer was addedand homogenized mechanically. After centrifugation, the supernatant wastaken and beta amyloid (IBL) concentration was measured. The amount ofAβ₄₀ expression in 1 mg protein was measured.

As a result, as shown in the following Table 10, it was confirmed thatthe production of beta amyloid was remarkably decreased byadministration of the herbal mixed extract. Compared with ComparativeExamples 1 and 2, which are sole extracts of mulberry and Poria cocospeel, and Comparative Examples 3 to 8, which are mixed extracts ofmulberry and Poria cocos peel, mulberry and Poria cocos peel 5:1 mixtureshowed a higher inhibitory effect on beta amyloid formation. Singleherbal extracts also showed beta amyloid-reducing activity. Among them,mulberry extract showed a relatively strong inhibitory effect onβ-amyloid formation, and beta-amyloid-reducing effect was increased inmixed extract of botanical raw materials when the weight ratio ofmulberry was high.

Donepezil (DPZ), an acetyl cholinesterase inhibitor used in Alzheimer'sdisease, did not affect beta amyloid production at a dose of 1 mg/kg.

TABLE 10 Normal animal model - Aβ₄₀ measurement Total weight Rawmaterial comparison amount (kg) Mulberry:Poria Aβ₄₀ Poria cocos concen-cocos peel weight Volume tration Mulberry peel ratio (mg/kg) (pg/mg)Vehicle 165.7 example 1 250 50 5:1 100 111.7 Comparative 200 — Mulberry100 120.1 Example 1 alone Comparative — 200 Poria cocos 100 145.6Example 2 peel alone Comparative 100 100 1:1 100 132.9 Example 3Comparative 150 50 3:1 100 127.8 Example 4 Comparative 200 20 10:1  100125.6 Example 5 Comparative 50 150 1:3 100 134.9 Example 6 Comparative40 200 1:5 100 134.6 Example 7 Comparative 20 200  1:10 100 141.2Example 8 Donepezil 1 168.8

<1-7> NGF (Nerve Growth Factor) Level Measurement

ELISA experiments were conducted to confirm the efficacy of the mixedextracts on NGF production, known to be a neuronal regeneration anddifferentiation activity. After the brains of normal animals inExperimental Example <1-1> were extracted, the hippocampal region wasseparated, and RIPA buffer was added thereto and mechanicallyhomogenized. For NGF measurement, a high salt high detergent buffercorresponding to the weight of each brain was added and mechanicallyhomogenized. After adding 10 μl of 4N HCl and allowing to stand for 15minutes, NGF bound to the receptor was dissociated, 4N NaOH was added,and the mixture was allowed to stand for another 15 minutes. Thesupernatant was taken by centrifugation and the NGF concentration in thebrain was measured using an NGF measurement kit (Millipore). The amountof NGF produced in 1 mg protein is shown in the table.

As a result, as shown in the following Table 11, it was confirmed thatNGF production was significantly increased by the administration of theherbal mixed extract. Compared with Comparative Examples 1 and 2, whichare sole extracts of mulberry and Poria cocos peel, and ComparativeExamples 3 to 8, which are mixed extracts of mulberry and Poria cocospeel, In Example 1 according to the present invention, a mixture ofmulberry and Poria cocos peel 5:1 showed higher NGF production promotingactivity. Single herbal extracts also stimulated the production of NGF.Among them, Poria cocos peel extract showed relatively strong promotingeffect on NGF production. In the mixed extract of botanical rawmaterials, when the weight ratio of Poria cocos peel was high, thepromoting effect of NGF production was large.

Donepezil (DPZ), an acetyl cholinesterase inhibitor used in Alzheimer'sdisease, did not affect NGF production at a dose of 1 mg/kg.

TABLE 11 Normal animal model - NGF measurement Raw material Total weightamount (kg) comparison NGF Poria Mulberry:Poria concen- cocos cocos peelVolume tration Mulberry peel weight ratio (mg/kg) (pg/mg) Vehicle 247.7example 1 250 50 5:1 100 395.7 Comparative 200 — Mulberry 100 256.0Example 1 alone Comparative — 200 Poria cocos 100 341.0 Example 2 peelalone Comparative 100 100 1:1 100 280.7 Example 3 Comparative 150 50 3:1100 304.0 Example 4 Comparative 200 20 10:1  100 311.0 Example 5Comparative 50 150 1:3 100 329.0 Example 6 Comparative 40 200 1:5 100347.3 Example 7 Comparative 20 200  1:10 100 360.3 Example 8 Donepezil 1258.0

<Experimental Example 2> Identification of Nerve Cell Protective Effectof Mulberry and Poria cocos Peel Mixture Extracts In Vitro

<2-1> Measurement of Acetyl Cholinesterase (AChE) Activity in Nerve CellLines

Acetyl cholinesterase inhibitors have been developed and used forvarious acetyl cholinesterase inhibitors because they enhance the memorycapacity and improve the dementia by activating the cholinergic neuronsby increasing the acetylcholine concentration in the ganglionneurotransmitter. The acetyl cholinesterase activity was measured toconfirm the acetyl cholinesterase inhibitory effect of the herbal mixedextract. SH-SY5Y cells, a neuronal cell line, were purchased from KCLB(Korean Cell Line Bank). SH-SY5Y cells (1×106 cells/well) wereinoculated on 6-well culture plates and cultured in DMEM/F12 (Dulbecco'smodified Eagle's medium) medium containing 10% fetal bovine serum for 48hours. After 5 days of differentiation using DMEM/F12 medium containing10 μM retinoic acid and 3% FBS, the Examples, the Comparative Example(10 μg/ml) and the control drug (donepezil 10 μM), Aβ42 was treated.Cell lysis was performed using RIPA buffer (150 mM NaCl, 0.5% TritonX-100, 50 mM Tris-HCl, pH 7.4, 25 mM NaF, 20 mM EGTA, 1 mM DTT, 1 mMNa3VO4, protease inhibitor cocktail), And protein content wasquantitated with BCA reagent purchased from Pierce. The acetylcholinesterase enzyme activity was determined by acetylcholine as asubstrate according to the Ellman method. The resulting thiocholineproduced by acetyl cholinesterase was reacted with DTNB and theresulting 5-thio-2-nitrobenzoate was determined by measuring theabsorbance change at 405 nm.

As a result, as shown in Table 12 below, it was confirmed that theacetyl cholinesterase activity increased by the beta amyloid treatmentwas normalized by the administration of the herbal mixed extract.Compared with Comparative Examples 1 and 2 which are the sole extractsof mulberry and Poria cocos peel and Comparative Examples 3 to 8 whichare mixed extracts of mulberry and Poria cocos peel, The mixture ofmulberry and Poria cocos peel 5:1 of Example 1 according to the presentinvention showed excellent acetyl cholinesterase activity inhibitoryactivity. The single herbal extracts of Comparative Examples 1 and 2exhibited slight acetyl cholinesterase inhibitory action, but bothsingle herbal extracts showed lower efficacy than the herbal mixedextracts. The inhibitory effect of herbal extracts was similar to thatof Donepezil (DPZ) 10 μM, which is an acetyl cholinesterase inhibitorand used in Alzheimer's disease.

TABLE 12 Neuronal cell line model - acetyl cholinesterase activityAcetyl Raw material Total weight cholines- amount (kg) comparison terasePoria Mulberry:Poria activity cocos cocos peel Volume (mU/mg Mulberrypeel weight ratio (μg/ml) protein) Normal 8.94 Vehicle 13.54 example 1250 50 5:1 10 5.64 Comparative 200 — Mulberry 10 9.84 Example 1 aloneComparative — 200 Poria cocos 10 7.23 Example 2 peel alone Comparative100 100 1:1 10 8.54 Example 3 Comparative 150 50 3:1 10 6.32 Example 4Comparative 200 20 10:1  10 7.32 Example 5 Comparative 50 150 1:3 108.11 Example 6 Comparative 40 200 1:5 10 8.32 Example 7 Comparative 20200  1:10 10 7.64 Example 8 Donepezil 10 (μM) 5.38

<2-2> Cell Protection Effect in Nerve Cell Line

The cytoprotective effect of herbal mixed extracts was measured using aMTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assayin a model that causes toxicity to nerve cell lines by artificiallyaggregated Aβ₄₂. SH-SY5Y cells (1×10⁴ cells/well) were inoculated into96-well culture plates and cultured in DMEM/F12 (Dulbecco's modifiedEagle's medium: nutrient mixture F-12) containing 10% fetal bovine serumfor 48 hours. After 5 days of differentiation using DMEM/F12 mediumcontaining 10 μM retinoic acid and 3% 1-BS, 6 hours after the treatmentof the Examples, the Comparative Example and the control drug (donepezil10 μM), Aβ₄₂ was treated for a total of 48 hours, and after 4 hours oftreatment with 2 mg/ml MTT, Formazone was dissolved in DMSO andabsorbance was measured at 590 nm.

As a result, it was confirmed that the cytotoxicity induced by betaamyloid treatment was inhibited by the herbal mixed extractadministration as shown in Table 13 below. Compared with ComparativeExamples 1 and 2 which are solely extracts of mulberry and Poria cocospeel and Comparative Examples 3 to 8 which are mixed extracts ofmulberry and Poria cocos peel, mulberry, Poria cocos peel 5:1 mixtureshowed excellent cytoprotective effect. Donepezil (DPZ), an acetylcholinesterase inhibitor used in Alzheimer's disease, showed a low levelof cytoprotective activity at a dose of 10 μM.

TABLE 13 Neuronal cell model - MTT measurement value MTT Raw materialTotal weight measure- amount (kg) comparison ment Poria Mulberry:Poriavalue cocos cocos peel Volume (% of Mulberry peel weight ratio (μg/ml)Normal) Normal 100.0 Vehicle 56.2 example 1 250 50 5:1 10 89.3Comparative 200 — Mulberry 10 64.3 Example 1 alone Comparative — 200Poria cocos 10 82.3 Example 2 peel alone Comparative 100 100 1:1 10 68.9Example 3 Comparative 150 50 3:1 10 72.3 Example 4 Comparative 200 2010:1  10 79.3 Example 5 Comparative 50 150 1:3 10 66.4 Example 6Comparative 40 200 1:5 10 68.9 Example 7 Comparative 20 200  1:10 1079.4 Example 8 Donepezil 10 (μM) 65.3

<2-3> Measurement of Phosphorylated Tau in Neuronal Cell Lines

Phosphorylated tau levels were measured in order to confirm theinhibitory effect of crude herbal extracts on tau hyper-phosphorylation.SH-SY5Y cells (1×106 cells/well) were seeded in 6-well culture platesand cultured in DMEM/F12 (Dulbecco's modified Eagle's medium) mediumcontaining 10% FBS (fetal bovine serum) for 48 hours. After 5 days ofdifferentiation using DMEM/F12 medium containing 10 μM retinoic acid and3% FBS, After 6 hours of treatment with the example, comparative example(10 μg/ml) and a control drug (donepezil 10 μM), Aβ₄₂ was treated andcultured for a total of 48 hours. After cell lysis using RIPA buffer,the protein content was quantitated with BCA reagent purchased fromPierce. The phosphorylated tau was measured using human Tau [pS199]enzyme-linked immunosorbent assay (ELISA) KIT purchased from Invitrogen.

As a result, it was confirmed that the increase of tau phosphorylationinduced by the beta amyloid treatment was suppressed by the herbal mixedextract as shown in Table 14 below. Compared with Comparative Examples 1and 2, which are sole extracts of mulberry and Poria cocos peel, andComparative Examples 3 to 8, which are mixed extracts of mulberry andPoria cocos peel, the tau phosphorylation inhibitory effect wasexcellent in the mixture of Example 1 mulberry and Poria cocos peel 5:1.In particular, the effect of inhibiting tau phosphorylation wassignificantly increased by the mixed extracts when compared to themulberry and Poria cocos peel alone extracts. Donepezil (DPZ), an acetylcholinesterase inhibitor used in Alzheimer's disease, did not show anyinhibitory effect on tau phosphorylation at a dose of 10 μM.

TABLE 14 Neuronal cell model - Tau phosphorylation measurement Totalweight Raw material comparison amount (kg) Mulberry:Poria Poria cocosPhosphorus cocos peel weight Volume tau pg/g Mulberry peel ratio (μg/ml)protein) Normal 1197 Vehicle 1684 example 1 250 50 5:1 10 864Comparative 200 — Mulberry alone 10 1579 Example 1 Comparative — 200Poria cocos peel 10 1021 Example 2 alone Comparative 100 100 1:1 10 1324Example 3 Comparative 150 50 3:1 10 1027 Example 4 Comparative 200 2010:1  10 1198 Example 5 Comparative 50 150 1:3 10 1318 Example 6Comparative 40 200 1:5 10 1225 Example 7 Comparative 20 200  1:10 101150 Example 8 Donepezil 10 (μM) 1699

<2-4> Nerve Growth Factor (NGF) Level Measurement in Nerve Cell Lines

NGF levels were measured in order to determine the mechanism ofcognitive improvement of the herbal mixed extracts. SH-SY5Y cells (1×106cells/well) were inoculated on 6-well culture plates and cultured inDMEM/F12 (Dulbecco's modified Eagle's medium) medium containing 10%fetal bovine serum for 48 hours. After 5 days of differentiation usingDMEM/F12 medium containing 10 μM retinoic acid and 3% FBS, to this, theexample, comparative example (10 μg/ml) and control drug (donepezil 10μM) were treated. Cells were lysed using RIPA buffer, and proteincontent was quantitated with BCA reagent purchased from Pierce. NGFlevels were measured using a beta-nerve growth factor humanenzyme-linked immunosorbent assay (ELISA) KIT purchased from Abcam.

As a result, as shown in Table 15, it was confirmed that the herbalmixed extract increased NGF production. At this time, beta amyloid didnot affect NGF production. As a result of comparing the action of NGFproduction of single herbal extracts, the efficacy of Poria cocos peelextract was high. Among the herbal mixed extracts, 5:1 combination wasthe most effective.

Donepezil (DPZ), which is used as an acetylcholinesterase inhibitor inAlzheimer's disease, did not show the effect of promoting NGF productionat a dose of 10 μM.

TABLE 15 Nerve cell model - NGF measurement Raw material Total weightamount (kg) comparison Poria Mulberry:Poria NGF cocos cocos peel Volume(pg/g Mulberry peel weight ratio (μg/ml) protein) Normal — — — — 438Vehicle — — — — 402 example 1 250 50 5:1 10 774 Comparative 200 —Mulberry 10 433 Example 1 alone Comparative — 200 Poria cocos 10 696Example 2 peel alone Comparative 100 100 1:1 10 453 Example 3Comparative 150 50 3:1 10 578 Example 4 Comparative 200 20 10:1  10 454Example 5 Comparative 50 150 1:3 10 488 Example 6 Comparative 40 200 1:510 533 Example 7 Comparative 20 200  1:10 10 655 Example 8 Donepezil — —— 10 (μM) 421

As described above, the extract of mulberry which is the material of theherbal mixed extract of the present invention appears to promote thesurvival of the neuron and to activate its function by removing betaamyloid which is considered to be a major cause of Alzheimer's dementia.Poria cocos peel extract, another material, is thought to be superior tonerve cell protection efficacy through promotion of NGF productionespecially, known as a neuronal protective factor. In addition, eachherbal extract did not exhibit high efficacy in inhibiting the acetylcholinesterase inhibitory effect, which is currently the major mechanismof action of drugs for Alzheimer's dementia. However, when they weremixed, the efficacy was increased. These results suggest that theextracts of mulberry and Poria cocos peel protect neurons andsynergistically increase their memory capacity by different mechanisms.Especially, when they are mixed at a weight ratio of 5:1, it isconsidered that the efficacy is excellent.

Therefore, the herbal mixed extract according to the present inventioninhibits the onset or progress of the disease by inhibiting beta amyloidproduction and tau phosphorylation, which cause degenerative braindiseases by causing neuronal cell death, and enhances memory ability byinhibiting acetylcholine esterase. It also promotes the production ofNGF, which promotes neuronal protection and neural differentiation.Therefore, it can be usefully used for prevention, amelioration andtreatment of progressive dementia due to gradual killing of neurons suchas Alzheimer's dementia and Creutzfeldt-Jakob disease, Huntington'sdisease, multiple sclerosis, Guilin-Barre syndrome, Parkinson's disease,Lou Gehrig's disease. It may also be useful for the prevention,amelioration and treatment of degenerative neurological disordersincluding posture and movement disorders, progressive ataxia, muscularatrophy and weakness, sensory and motor disorders and so on.

Hereinafter, a preparation example of a composition containing a crudedrug extract of Example 1 of the present invention will be described.However, it should be understood that the present invention is notintended to be limited thereto but is specifically described.

Preparation Example 1: Preparation of Injections

Example 1 Extract 100 mg  Sodium Metabisulfite 3.0 mg Methylparaben 0.8mg Propylparaben 0.1 mg Sterilized distilled water for injection Properamount

The above ingredients are mixed and made into 2 ml by a usual injectionpreparation method, after that filled in a 2 ml ampoule and sterilizedto prepare an injection.

Preparation Example 2: Preparation of Tablets

Example 1 Extract 200 mg Lactose 100 mg Starch 100 mg Magnesium stearateProper amount

The above components are mixed and tablets are prepared by tabletingaccording to the conventional tablet preparation method.

Preparation Example 3: Preparation of Capsules

Example 1 Extract 100 mg Lactose  50 mg Starch 100 mg Talc  2 mgMagnesium stearate Proper amount

The above components are mixed and filled in gelatin capsules accordingto the conventional preparation method of capsules to prepare capsules.

Preparation Example 4: Preparation of Liquid Agent

Example 1 Ethanol extract 1000 mg sugar 20 g High Fructose Corn 20 gLemon incense Proper amount After adding purified water, the totalliquid 100 m

volume

The above components are mixed according to the usual method forproducing a liquid preparation, filled in 100 ml of a brown bottle andsterilized to prepare a liquid preparation.

1.-15. (canceled)
 16. A method of increasing the production of nervegrowth factor (NGF) in a subject in need thereof, comprisingadministering a composition comprising an extract of a mixture whichcomprises (i) mulberry and (ii) Poria cocos peel to the subject.
 17. Themethod of claim 16, wherein the weight ratio of the mulberry and Poriacocos peel is 4 to 7:1.
 18. The method of claim 16, wherein the weightratio of the mulberry and Poria cocos peel is 5:1.
 19. The method ofclaim 16, wherein the composition improves cognitive abilities in thesubject.
 20. The method of claim 16, wherein the composition enhancesmemory in the subject.
 21. A method of treating or amelioratingAlzheimer's Dementia in a subject in need thereof, comprisingadministering a composition comprising an extract of a mixture whichcomprises (i) mulberry and (ii) Poria cocos peel to the subject.
 22. Themethod of claim 21, wherein the weight ratio of the mulberry and Poriacocos peel is 4 to 7:1.
 23. The method of claim 21, wherein the weightratio of the mulberry and Poria cocos peel is 5:1.
 24. A method ofdecreasing the production of beta amyloid, reducing acetylcholinesterase activity, and/or reducing tau phosphoryation in a subjectin need thereof comprising administering an extract of a mixture whichcomprises (i) mulberry and (ii) Poria cocos peel to the subject.
 25. Themethod of claim 24, wherein the composition improves cognitive abilitiesin the subject.
 26. The method of claim 24, wherein the compositionenhances memory in the subject.
 27. The method of claim 26, wherein theweight ratio of the mulberry and Poria cocos peel is 4 to 7:1.
 28. Themethod of claim 26, wherein the weight ratio of the mulberry and Poriacocos peel is 5:1.